(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol and its uses

ABSTRACT

This invention discloses (20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol and pharmaceutical uses therefor. This compound exhibits pronounced activity in arresting the proliferation of undifferentiated cells and inducing their differentiation to the monocyte thus evidencing use as an anti-cancer agent and for the treatment of skin diseases such as psoriasis as well as skin conditions such as wrinkles, slack skin, dry skin and insufficient sebum secretion. This compound also has little, if any, calcemic activity and therefore may be used to treat immune disorders in humans as well as renal osteodystrophy.

BACKGROUND OF THE INVENTION

This invention relates to vitamin D compounds, and more particularly to(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol and itspharmaceutical uses.

The natural hormone, 1α,25-dihydroxyvitamin D₃ and its analog inergosterol series, i.e. 1α,25-dihydroxyvitamin D₂ are known to be highlypotent regulators of calcium homeostasis in animals and humans, andtheir activity in cellular differentiation has also been established,Ostrem et al., Proc. Natl. Acad. Sci. USA, 84, 2610 (1987). Manystructural analogs of these metabolites have been prepared and tested,including 1α-hydroxyvitamin D₃, 1α-hydroxyvitamin D₂, various side chainhomologated vitamins and fluorinated analogs. Some of these compoundsexhibit an interesting separation of activities in cell differentiationand calcium regulation. This difference in activity may be useful in thetreatment of a variety of diseases as renal osteodystrophy, vitaminD-resistant rickets, osteoporosis, psoriasis, and certain malignancies.

Recently, a new class of vitamin D analogs has been discovered, i.e. theso called 19-nor-vitamin D compounds, which are characterized by thereplacement of the A-ring exocyclic methylene group (carbon 19), typicalof the vitamin D system, by two hydrogen atoms. Biological testing ofsuch 19-nor-analogs (e.g., 1α,25-dihydroxy-19-nor-vitamin D₃) revealed aselective activity profile with high potency in inducing cellulardifferentiation, and very low calcium mobilizing activity. Thus, thesecompounds are potentially useful as therapeutic agents for the treatmentof malignancies, or the treatment of various skin disorders. Twodifferent methods of synthesis of such 19-nor-vitamin D analogs havebeen described (Perlman et al., Tetrahedron Lett. 31, 1823 (1990);Perlman et al., Tetrahedron Lett. 32, 7663 (1991), and DeLuca et al.,U.S. Pat. No. 5,086,191).

In U.S. Pat. No. 4,666,634, 2β-hydroxy and alkoxy (e.g., ED-71) analogsof 1α,25-dihydroxyvitamin D₃ have been described and examined by Chugaigroup as potential drugs for osteoporosis and as antitumor agents. Seealso Okano et al., Biochem. Biophys. Res. Commun. 163, 1444 (1989).Other 2-substituted (with hydroxyalkyl, e.g., ED-120, and fluoroalkylgroups) A-ring analogs of 1α25-dihydroxyvitamin D₃ have also beenprepared and tested (Miyamoto et al., Chem. Pharm. Bull. 41, 1111(1993); Nishii et al., Osteoporosis Int. Suppl. 1, 190 (1993); Posner etal., J. Org. Chem. 59, 7855 (1994), and J. Org. Chem. 60, 4617 (1995)).

Recently, 2-substituted analogs of 1α,25-dihydroxy-19-nor-vitamin D₃have also been synthesized, i.e. compounds substituted at 2-positionwith hydroxy or alkoxy groups (DeLuca et al., U.S. Pat. No. 5,536,713),with 2-alkyl groups (DeLuca et al U.S. Pat. No. 5,945,410), and with2-alkylidene groups (DeLuca et al U.S. Pat. No. 5,843,928), whichexhibit interesting and selective activity profiles. All these studiesindicate that binding sites in vitamin D receptors can accommodatedifferent substituents at C-2 in the synthesized vitamin D analogs.

In a continuing effort to explore the 19-nor class of pharmacologicallyimportant vitamin D compounds, an analog which is characterized by thepresence of a methylene substituent at the carbon 2 (C-2) has beensynthesized and tested. Of particular interest is the analog which ischaracterized by a hydroxyl group at carbon 1 and a shortened side chainattached to carbon 20, i.e.(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol. Thisvitamin D analog seemed an interesting target because the relativelysmall methylene group at C-2 should not interfere with binding to thevitamin D receptor. Moreover, molecular mechanics studies performed onthe model 1α-hydroxy-2-methylene-19-nor-vitamins indicate that suchmolecular modification does not change substantially the conformation ofthe cyclohexanediol ring A. However, introduction of the 2-methylenegroup into 19-nor-vitamin D carbon skeleton changes the character of its1α- and 3β- A-ring hydroxyls. They are both now in the allylicpositions, similarly, as 1α-hydroxyl group (crucial for biologicalactivity) in the molecule of the natural hormone, 1α,25-(OH)₂D₃.

SUMMARY OF THE INVENTION

The present invention is directed toward(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol, itsbiological activity, and various pharmaceutical uses for this compound.

Structurally this 19-nor analog is characterized by the general formulaI shown below:

The above compound exhibits a desired, and highly advantageous, patternof biological activity. This compound is characterized by relativelyhigh binding to vitamin D receptors, but very low intestinal calciumtransport activity, as compared to that of 1α,25-dihydroxyvitamin D₃,and has very low ability to mobilize calcium from bone, as compared to1α,25-dihydroxyvitamin D₃. Hence, this compound can be characterized ashaving little, if any, calcemic activity. Thus, it may be useful as atherapy for suppression of secondary hyperparathyroidism of renalosteodystrophy.

The compound of the invention has also been discovered to be especiallysuited for treatment and prophylaxis of human disorders which arecharacterized by an imbalance in the immune system, e.g. in autoimmunediseases, including multiple sclerosis, lupis, diabetes mellitus, hostversus graft reaction, and rejection of organ transplants; andadditionally for the treatment of inflammatory diseases, such asrheumatoid arthritis, asthma, and inflammatory bowel diseases such asceliac disease and croans disease, as well as the improvement of bonefracture healing and improved bone grafts. Acne, alopecia andhypertension are other conditions which may be treated with the compoundof the invention.

The above compound is also characterized by relatively high celldifferentiation activity. Thus, this compound also provides atherapeutic agent for the treatment of psoriasis, or as an anti-canceragent, especially against leukemia, colon cancer, breast cancer andprostate cancer. In addition, due to its relatively high celldifferentiation activity, this compound provides a therapeutic agent forthe treatment of various skin conditions including wrinkles, lack ofadequate dermal hydration, i.e. dry skin, lack of adequate skinfirmness, i.e. slack skin, and insufficient sebum secretion. Use of thiscompound thus not only results in moisturizing of skin but also improvesthe barrier function of skin.

The compound may be present in a composition to treat the above-noteddiseases and disorders in an amount from about 0.01 μg/gm to about 100μg/gm of the composition, and may be administered topically,transdermally, orally or parenterally in dosages of from about 0.01μg/day to about 100 μg/day.

BRIEF DESCRIPTION OF THE DRAWINGS

FIGS. 1A-1E illustrate the structures of the compounds described andtested herein, namely, 1α,25-dihydroxy-19-nor-vitamin D₃, hereinafter1,25(OH)₂19-nor-D₃ (FIG. 1A); the native hormone 1α,25-dihydroxyvitaminD₃, hereinafter 1,25(OH)₂D₃ (FIG. 1B);1α-hydroxy-2-methylene-19-nor-pregnacalciferol, hereinafter 2-Mpregna(FIG. 1C); (20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol,hereinafter 2-MbisP (FIG. 1D); and1α-hydroxy-2-methylene-19-nor-homopregnacalciferol, hereinafter 2-MP(FIG. 1E);

FIG. 2 is a graph illustrating the relative activity of 1,25(OH)₂19-nor-D₃, 2-Mpregna, 2-MbisP, 2-MP and 1,25(OH)₂D₃ to compete forbinding with [³H]-1,25-(OH)₂-D₃ to the full-length recombinant ratvitamin D receptor;

FIG. 3 is a graph illustrating the percent HL-60 cell differentiation asa function of the concentration of 1,25(OH)₂ 19-nor-D₃, 2-Mpregna,2-MbisP, 2-MP and 1,25(OH)₂D₃;

FIGS. 4A-4C are bar graphs illustrating the bone calcium mobilizationactivity of 1,25(OH)₂D₃ and 1,25(OH)₂ 19-nor-D₃ as compared to 2-MbisP(FIG. 4A), 2-Mpregna (FIG. 4B), and 2-MP (FIG. 4C);

FIGS. 5A-5C are bar graphs illustrating the intestinal calcium transportactivity of 1,25(OH)₂D₃ and 1,25(OH)₂ 19-nor-D₃ as compared to 2-MbisP(FIG. 4A), 2-Mpregna (FIG. 4B), and 2-MP (FIG. 4C); and

FIG. 6 is a bar graph illustrating blood serum calcium levels in femalerats after treatment with chronic doses of 1,25(OH)₂D₃ and 1,25(OH)₂19-nor-D₃ as compared to 2-MbisP (FIG. 6A), 2-Mpregna (FIG. 6B), and2-MP (FIG. 6C).

DETAILED DESCRIPTION OF THE INVENTION

(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol (referred toherein as 2-MbisP) was synthesized and tested. Structurally, this 19-noranalog is characterized by the general formula I previously illustratedherein.

The preparation of(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol having thebasic structure I can be accomplished by a common general method, i.e.the condensation of a bicyclic Windaus-Grundmann type ketone II with theallylic phosphine oxide III to the corresponding2-methylene-19-nor-vitamin D analog IV followed by deprotection at C-1and C-3 in the latter compound:

In the structures II, III, and IV groups Y₁ and Y₂ arehydroxy-protecting groups, it being also understood that anyfunctionalities that might be sensitive, or that interfere with thecondensation reaction, be suitably protected as is well-known in theart. The process shown above represents an application of the convergentsynthesis concept, which has been applied effectively for thepreparation of vitamin D compounds [e.g. Lythgoe et al., J. Chem. Soc.Perkin Trans. I, 590 (1978); Lythgoe, Chem. Soc. Rev. 9, 449 (1983); Tohet al., J. Org. Chem. 48, 1414 (1983); Baggiolini et al., J. Org. Chem.51, 3098 (1986); Sardina et al., J. Org. Chem. 51, 1264 (1986); J. Org.Chem. 51, 1269 (1986); DeLuca et al., U.S. Pat. No. 5,086,191; DeLuca etal., U.S. Pat. No. 5,536,713].

Hydrindanones of the general structure II are known, or can be preparedby known methods.

For the preparation of the required phosphine oxides of generalstructure III, a synthetic route has been developed starting from amethyl quinicate derivative which is easily obtained from commercial(1R,3R,4S,5R)-(−)-quinic acid as described by Perlman et al.,Tetrahedron Lett. 32, 7663 (1991) and DeLuca et al., U.S. Pat. No.5,086,191.

The overall process of the synthesis of compound I is illustrated anddescribed more completely in U.S. Pat. No. 5,843,928 entitled“2-Alkylidene-19-Nor-Vitamin D Compounds” the specification of which isspecifically incorporated herein by reference.

Biological Activity of(20S)-1α-Hydroxy-2-Methylene-19-Nor-Bishomopregnacalciferol

The introduction of a methylene group to the 2-position of1α-hydroxy-19-nor-pregnacalciferol had little or no effect on binding tothe porcine intestinal vitamin D receptor, as compared to1α,25-dihydroxyvitamin D₃. This compound bound equally well to theporcine receptor as compared to the standard 1,25-(OH)₂D₃ (FIG. 2). Itmight be expected from these results that this compound would haveequivalent biological activity. Surprisingly, however, the 2-methylenesubstitution in 2-MbisP produced a highly selective analog with uniquebiological activity.

FIG. 4B shows that 2-MbisP has very little activity as compared to thatof 1,25-dihydroxyvitamin D₃ (1,25(OH)₂D₃), the natural hormone, instimulating intestinal calcium transport.

FIG. 5B demonstrates that 2-MbisP has very little bone calciummobilization activity, as compared to 1,25(OH)₂D₃.

FIGS. 4B and 5B thus illustrate that 2-MbisP may be characterized ashaving little, if any, calcemic activity.

FIG. 3 illustrates that 2-MbisP is almost as potent as 1,25(OH)₂D₃ onHL-60 differentiation, making it an excellent candidate for thetreatment of psoriasis and cancer, especially against leukemia, coloncancer, breast cancer and prostate cancer. In addition, due to itsrelatively high cell differentiation activity, this compound provides atherapeutic agent for the treatment of various skin conditions includingwrinkles, lack of adequate dermal hydration, i.e. dry skin, lack ofadequate skin firmness, i.e. slack skin, and insufficient sebumsecretion. Use of this compound thus not only results in moisturizing ofskin but also improves the barrier function of skin.

FIG. 6 shows an analysis of serum calcium in rats after administrationof chronic doses of 2-MbisP. These data provide further support for thedata in FIGS. 4B and 5B that 2-MbisP has very little calcemic activityand thus a relatively low risk of producing hypercalcemia at recommendeddoses.

The battery of in vitro and in vivo assays described in Sicinski et al(J. Med. Chem. 41, 4662-4674, 1998) were used to compare the biologicalactivities of the compounds, 2-Mpregna, 2-MbisP and 2-MP, with 1,25(OH)₂19-nor-D₃, and 1,25(OH)₂D₃, the native vitamin D hormone.

The differentiation of HL-60 promyelocytic into monocytes was determinedas described by Ostrem et al (J. Biol. Chem. 262, 14164-14171, 1987).

Interpretation of Data

VDR binding and HL60 cell differentiation. 2-MbisP, 2-Mpregna and 2-MPare nearly equivalent (Ki=0.3, 0.6 and 0.3 nM for 2-Mpregna, 2-MbisP and2-MP, respectively) in their ability to compete with [³H]-1,25(OH)₂D₃for binding to the full-length recombinant rat vitamin D receptor (FIG.2). Furthermore, the competition binding activity of these threecompounds is similar to that of 1,25(OH)₂ 19-nor-D₃ (Ki=0.2 nM), as wellas the native hormone, 1,25(OH)₂D₃ (Ki=0.1 nM). There is littledifference between any of these five compounds in their ability(efficacy or potency) to promote HL60 differentiation with the possibleexception of 2-Mpregna (EC₅₀=17 nM), which is slightly less potent than2-MbisP (EC₅₀=7 nM), 2-MP (EC₅₀=6 nM), 1,25(OH)₂ 19-nor-D₃ (EC₅₀=4 nM)and 1,25(OH)₂D₃ (EC₅₀=5 nM) (See FIG. 3). This result suggests that2-MbisP will be very effective in psoriasis because it has directcellular activity in causing cell differentiation and in suppressingcell growth. It also indicates that it will have significant activity asan anti-cancer agent, especially against leukemia, colon cancer, breastcancer and prostate cancer, as well as against skin conditions such asdry skin (lack of dermal hydration), undue skin slackness (insufficientskin firmness), insufficient sebum secretion and wrinkles.

Calcium mobilization from bone and intestinal calcium absorption invitamin D-deficient animals. Using vitamin D-deficient rats on a lowcalcium diet (0.02%), the activities of these compounds in intestine andbone were tested. As expected, the native hormone (1,25(OH)₂D₃)increased serum calcium levels at all dosages (FIG. 4). FIG. 4 alsoshows that 2-MbisP, 2-Mpregna and 2-MP have little, if any, activity inmobilizing calcium from bone. Administration of 2-MbisP, 2-Mpregna, 2-MPor 1,25(OH)₂ 19-nor-D₃ at 260 pmol/day for 7 days did not result inmobilization of bone calcium, and increasing the amount of 2-MbisP,2-Mpregna and 2-MP to 1300 pmol/day (5-fold) was without effect. Similarfindings were obtained with 1,25(OH)₂ 19-nor-D₃.

Intestinal calcium transport was evaluated in the same groups of animalsusing the everted gut sac method (FIG. 5). These results show that2-MbisP and 2-Mpregna do not promote intestinal calcium transport whenadministered at either 260 or 1300 pmol/day, whereas 1,25(OH)₂D₃promotes a significant increase at the 260 pmol/day dose. In contrast to2-MbisP and 2-Mpregna, 2-MP showed calcium transport activity equivalentto 1,25(OH)₂D₃ at this and the 5-fold higher concentration but higherdoses actually decreased this activity. As shown in FIG. 4, 1,25(OH)₂19-nor-D₃, like the 2-MbisP and 2-Mpregna derivatives, is devoid ofintestinal calcium transport activity.

Serum calcium response in vitamin D-sufficient animals on a normalcalcium diet. The desirability of 2-MbisP, 2-Mpregna and 2-MP isexemplified by their inability to produce hypercalcemia in normalanimals compared to 1,25(OH)₂ 19-nor-D₃, and 1,25(OH)₂D₃. In theexperiment shown in FIG. 6, normal, female rats were given 2500 pmol/dayfor 7 days of 1,25(OH)₂D₃, or 5000 pmol/day of 2-MbisP, 2-Mpregna, 2-MPor 1,25(OH)₂ 19-nor-D₃. The doses were administered to two separategroups of animals by either the oral or intraperitoneal route, and serumcalcium levels were assessed 4 hours after the last dose. Animalsreceiving 1,25(OH)₂D₃ by either route exhibited hypercalcemia, somesevere enough to require euthanasia. Likewise, 1,25(OH)₂ 19-nor-D₃produced frank hypercalcemia. In contrast, no increase in serum calciumwas seen in any of the animals receiving the compounds, 2-MP, 2-MbisP or2-Mpregna.

These results illustrate that 2-MbisP is an excellent candidate fornumerous human therapies and that it may be useful in a number ofcircumstances such as autoimmune diseases, cancer, and psoriasis.2-MbisP is an excellent candidate for treating psoriasis because: (1) ithas significant VDR binding and cellular differentiation activity; (2)it is devoid of hypercalcemic liability unlike 1,25(OH)₂ 19-nor-D₃ and1,25(OH)₂D₃; and (3) it is easily synthesized. Since 2-MbisP hassignificant binding activity to the vitamin D receptor, but has littleability to raise blood serum calcium, it may also be useful for thetreatment of renal osteodystrophy.

For treatment purposes, the compound of this invention defined byformula I may be formulated for pharmaceutical applications as asolution in innocuous solvents, or as an emulsion, suspension ordispersion in suitable solvents or carriers, or as pills, tablets orcapsules, together with solid carriers, according to conventionalmethods known in the art. Any such formulations may also contain otherpharmaceutically-acceptable and non-toxic excipients such asstabilizers, anti-oxidants, binders, coloring agents or emulsifying ortaste-modifying agents.

The compound may be administered orally, topically, parenterally ortransdermally. The compound is advantageously administered by injectionor by intravenous infusion or suitable sterile solutions, or in the formof liquid or solid doses via the alimentary canal, or in the form ofcreams, ointments, patches, or similar vehicles suitable for transdermalapplications. Doses of from 0.01 μg to 100 μg per day of the compoundsare appropriate for treatment purposes, such doses being adjustedaccording to the disease to be treated, its severity and the response ofthe subject as is well understood in the art. Since the compoundexhibits specificity of action, each may be suitably administered alone,or together with graded doses of another active vitamin D compound—e.g.1α-hydroxyvitamin D₂ or D₃, or 1α,25-dihydroxyvitamin D₃—in situationswhere different degrees of bone mineral mobilization and calciumtransport stimulation is found to be advantageous.

Compositions for use in the above-mentioned treatments comprise aneffective amount of the(20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferol as definedby the above formula I as the active ingredient, and a suitable carrier.An effective amount of such compound for use in accordance with thisinvention is from about 0.01 μg to about 100 μg per gm of composition,and may be administered topically, transdermally, orally or parenterallyin dosages of from about 0.01 μg/day to about 100 μg/day.

The compound may be formulated as creams, lotions, ointments, topicalpatches, pills, capsules or tablets, or in liquid form as solutions,emulsions, dispersions, or suspensions in pharmaceutically innocuous andacceptable solvent or oils, and such preparations may contain inaddition other pharmaceutically innocuous or beneficial components, suchas stabilizers, antioxidants, emulsifiers, coloring agents, binders ortaste-modifying agents.

The compound is advantageously administered in amounts sufficient toeffect the differentiation of promyelocytes to normal macrophages.Dosages as described above are suitable, it being understood that theamounts given are to be adjusted in accordance with the severity of thedisease, and the condition and response of the subject as is wellunderstood in the art.

The formulations of the present invention comprise an active ingredientin association with a pharmaceutically acceptable carrier therefore andoptionally other therapeutic ingredients. The carrier must be“acceptable” in the sense of being compatible with the other ingredientsof the formulations and not deleterious to the recipient thereof.

Formulations of the present invention suitable for oral administrationmay be in the form of discrete units as capsules, sachets, tablets orlozenges, each containing a predetermined amount of the activeingredient; in the form of a powder or granules; in the form of asolution or a suspension in an aqueous liquid or non-aqueous liquid; orin the form of an oil-in-water emulsion or a water-in-oil emulsion.

Formulations for rectal administration may be in the form of asuppository incorporating the active ingredient and carrier such ascocoa butter, or in the form of an enema.

Formulations suitable for parenteral administration convenientlycomprise a sterile oily or aqueous preparation of the active ingredientwhich is preferably isotonic with the blood of the recipient.

Formulations suitable for topical administration include liquid orsemi-liquid preparations such as liniments, lotions, applicants,oil-in-water or water-in-oil emulsions such as creams, ointments orpastes; or solutions or suspensions such as drops; or as sprays.

For asthma treatment, inhalation of powder, self-propelling or sprayformulations, dispensed with a spray can, a nebulizer or an atomizer canbe used. The formulations, when dispensed, preferably have a particlesize in the range of 10 to 100μ.

The formulations may conveniently be presented in dosage unit form andmay be prepared by any of the methods well known in the art of pharmacy.By the term “dosage unit” is meant a unitary, i.e. a single dose whichis capable of being administered to a patient as a physically andchemically stable unit dose comprising either the active ingredient assuch or a mixture of it with solid or liquid pharmaceutical diluents orcarriers.

We claim:
 1. (20S)-1α-hydroxy-2-methylene-19-nor-bishomopregnacalciferolhaving the formula: